冠心病相关miR-206启动子区转录因子的预测与鉴定

Prediction and identification of transcription factor of coronary heart disease associated hsa-miR-206 promoter

目的预测并初步鉴定转录因子对hsa-miR-206启动子区的靶向结合,以进一步研究其转录调控机制。方法通过生物信息学软件预测Myo D在hsa-miR-206启动子区有结合位点,分别构建p GL3-miR206 promoter萤光素酶报告载体和p EGFP-N2-Myo D表达质粒,分组共转染于He La细胞,应用双萤光素酶报告基因法检测定萤光素酶活性。结果生物信息学软件预测hsa-miR-206启动子区有2个转录因子Myo D结合位点。构建的2个载体经电泳及测序证实无误。双萤光素酶报告基因法检测显示,与对照组相比,共转染p GL3-Basic-miR206 promoter荧光素酶报告载体和p EGFP-N2-Myo D表达质粒实验组的荧光素酶活性显著降低,差异有统计学意义(P<0.05)。结论 Myo D能在hsa-miR-206启动子区靶向结合,是为hsa-miR-206上游转录因子,为后续研究miR-206的转录调控机制提供了一定的实验基础。

Objective To predict and analyze the transcription factor binding site of hsa- miR- 206 promoter,so as to further study the transcriptional regulation mechanism of miR- 206. Methods The binding sites of Myo D in hsa- miR- 206 promoter was obtained by bioinformatic software. Luciferase reporter gene vector of p GL3- miR206 promoter and p EGFP- N2- Myo D expression plasmid were constructed. After He La cells being transfected by different vectors,the dual- luciferase reporter assay system was used to detect the luciferase activity. Results Two binding sites of Myo D in hsa- miR- 206 promoter sequence were predicted by bioinformatic software. And the 2 vectors were confirmed with electrophoresis and sequencing. The dual- luciferase reporters gene assay system showed that compared with the control group,luciferase activity of He La cell transfected with p GL3- miR206 promoter vector and p EGFP- N2- Myo D vector decreased,with the differences statistically significant（ P 0. 05）. Conclusion Myo D can specifically target to miR- 206 promoter,which is the upstream transcription factor for hsa- mir- 206,and can provide a certain experimental basis for the follow- up study of the transcriptional regulation mechanism of miR- 206.